The present invention relates to immobilized garlic alliinase in its biologically active form, and the use of said immobilized alliinase in a method for the manufacture of substantially purified allicin for use as active ingredient of pharmaceutical compositions and as food additive.
Garlic and onions are members of the lilly family. Many medical properties have been ascribed to garlic and onions and they have been used in folk medicine for thousands of years.
A large spectrum of medical properties has been ascribed to garlic, Allium sativum, (Block, 1985). In moderm times the interest in the therapeutic properties of garlic has been resumed, and it is object of an increasing number of biochemical and clinical studies.
Garlic preparations are commercially available in the form of garlic oil, extracts, pills or tablets. Usually the preparation procedures of such garlic preparations are unknown, and the composition and amount of their active ingredients are not defined.
The active principles present in garlic have been found to be mainly sulfur-containing compounds. The principal component of a colorless oil obtained from steam distillates of garlic extracts was shown to be an unusual sulfur compound of formula C6H10S2O, named allicin (thio-2-propene-1-sulfinic acid S-allyl ester) (Cavallito et al., 1944). Allicin was found to be a chemically unstable, colorless liquid that is thought to be responsible for both the odor and much of the biological activity of garlic.
Although allicin is responsible for the smell of garlic, a garlic bulb exhibits little or no odor until it is cut or crushed. The intact garlic clove does not contain allicin but rather its odorless precursor alliin (+)(S-allyl-L-cysteine sulfoxide) that is converted to allicin, pyruvate and ammonia by a C-S-lyase present in the garlic plant termed alliin lyase or alliinase [E.C. 4.4.1.4.] (Stoll and Seebeck, 1949). Alliin and alliinase are found in different compartments of the garlic clove. The cutting or crushing of the clove enables the enzyme to come into contact with the precursor of allicin.
Studies performed with garlic preparations confirmed some of the medical activities attributed to garlic. Thus garlic juice was shown to inhibit the growth of bacteria of the genera Staphylococcus, Streptococcus, Vibrio and Bacillus and of zoopathogenic fungi and many strains of yeast, including Candida albicans (Block, 1985; Appleton and Tansey, 1975; Barone and Tansey, 1977), and allicin was shown to exhibit antibacterial, antifungal and antiamebic activity (Cavallito et al., 1944; Barone and Tansey, 1977; Mirelman et al., 1987).
In the last few years, many studies reported the beneficial effects of garlic on cardiovascular risk factors, mainly hyperlipidemia and thrombogenesis in animals and in humans. The results of the administration of fresh garlic, etheric extracts, or its active component allicin were consistent: garlic induces an increase in fibrinolytic activity (Bordia et al., 1977; Kieswetter et al., 1990); inhibits platelet aggregation (Makheja and Bailey, 1990); and improves lipid profile including reduction of serum cholesterol levels (Bordia and Verma, 1980; Bordia et al., 1975; Knipschild and Ter-Riet, 1989; Augusti and Mathew, 1974).
These studies demonstrated a very impressive effect of garlic, but most studies were limited by several factors such as lack of controlled methods and suitable double-blind studies and use of preparations with unknown amount and chemical identification of the active ingredient.
Allicin was shown to exhibit the beneficial properties ascribed to garlic and thus it would be useful to produce allicin in controlled and known amounts for use as the active ingredient of pharmaceutical compositions. However, allicin is a very labile and volatile compound when exposed to air and the methods known today for its preparation are not satisfactory. The chemical synthesis involves many steps and is complicated, laborious, expensive, and very inefficient. The enzymatic method seems to be more attractive, however alliinase is a so-called xe2x80x9csuicidal enzymexe2x80x9d that is rapidly and irreversibly inactivated by its own reaction product, allicin. Therefore a few minutes incubation of alliinase with the substrate alliin or its product, allicin, leads to a biologically inactive enzyme after one or a very limited number of cycles.
In order to overcome the above mentioned obstacles, the present invention provides alliinase in a form that is not inactivated by allicin. Thus, alliinase according to the invention can be used for the continuous preparation of allicin without losing its biological activity.
In one aspect, the present invention provides the garlic enzyme alliinase in an enzymatically active, immobilized form wherein the enzyme is chemically, physically or biologically immobilized on a support carrier.
Chemical immobilization according to the invention may be carried out by covalently binding the enzyme to a carrier selected from the group consisting of organic natural and synthetic polymers and inorganic carriers. Physical immobilization may be effected by entrapment of the enzyme within a polymer matrix or membrane, or its microencapsulation within semipermeable polymer membranes. Biological immobilization may be carried out by either binding biotinylated alliinase to an avidin-containing column or binding a cellulose binding domain (CBD) protein-modified alliinase to a cellulose column.
In another aspect, the invention provides a column comprising a chemically, physically or biologically immobilized garlic alliinase and a method for continuous production of substantially pure allicin using said column to which the substrate alliin is added.
By a further aspect, the present invention provides the use of substantially pure allicin produced according to the method of the invention for the preparation of pharmaceutical compositions for the treatment of viral, bacterial, fungal and parasitic infections, high levels of cholesterol and blood lipids, high blood pressure and thrombosis.
In yet another aspect the invention provides substantially pure allicin to be used as food additive or condiment.